Cartridge, kit and method for processing biological samples and manipulating liquids having biological samples

ABSTRACT

Cartridge has a container with at least one well having a channel from a well opening to a container base side, protrusions on the container base side, and a flat polymer film with a hydrophobic upper surface kept at a distance from the base side by the protrusions. The container and film are reversibly attachable to a liquid droplet manipulation instrument so the lower surface of the film abuts at least one electrode array of the instrument. The container enables displacement of a liquid droplet from a well through the channel onto the hydrophobic upper surface and above the electrode array. The instrument has a control unit with a voltage control and an electrode selector for individually selecting each electrode of the electrode array and for providing the selected electrode with a voltage to controlling guided movement of a liquid droplet by electrowetting. A kit and method are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of the patent application Ser. No.13/139,647 filed on Aug. 23, 2011, now U.S. Pat. No. ______, as 371national phase filing of the international application no.PCT/EP09/67240 filed Dec. 16, 2009, which claims priority on provisionalpatent application No. 61/138,294 filed Dec. 17, 2008, which are allincorporated herein by reference, and which also claims priority onSwiss patent application no. 01979/08 filed Dec. 17, 2008, whichpriority claims are both repeated here.

FIELD OF TECHNOLOGY

The present invention relates to a biological sample processing systemcomprising a container for large volume processing, a flat polymer filmand a liquid droplet manipulation instrument. The invention furtherrelates to a liquid droplet manipulation instrument comprising anelectrode array supported by a substrate, and a central processing unitfor controlling the selection of individual electrodes providing themwith individual voltage pulses for manipulating liquid droplets byelectrowetting. Preferably, this liquid droplet manipulation instrumentis accomplished to receive a flat polymer film as well as such acontainer for large volume processing.

The analysis of biological material such as tissue samples ormicroorganisms, in particular nucleic acids or proteins, is wellestablished in various fields, especially in the field of scientificresearch, pharmacological screening or forensic sciences. Adequatemethods have been developed for different purposes, each methodrequiring a special set of reaction reagents and devices for theperformance of the respective method. However it remains a challenge toadopt existing analysis procedures to the different conditions andrequirements present in each field. For example in criminal forensics, arelatively small amount of material to be analyzed is usually available.Additionally, the quality of such material can be rather low, placingadditional challenges on the involved personal. Thus, the proceduresneed to be specifically adapted to these conditions. On the other hand,for laboratory diagnostic procedures the biological material is usuallyavailable in sufficient amounts, but the required methods are to beadopted individually depending on the underlying question to be solved.

For the first steps of the analysis of biological material, there aremethods required, which are well known in the art. Material of interestis collected e.g. from a crime scene (in criminal forensics) or from apatient (for diagnostic purposes). Such materials can be tissue samples(such as oral mucosa cells, hair follicles) or bodily fluids (such asblood, sputum, etc.). This starting material then requires furtherprocessing to make nucleic acids or proteins available for the analysis.Typically, a lysis step is initially applied for these purposes,involving for example the application of heat, a certain enzymaticactivity, and/or the application of specific chemicals. The cell lysisis followed by a purification of the nucleic acid or protein of interestfrom the additional cellular material. In the case where the nucleicacid is to be analyzed, an amplification step might be advisable toincrease the sample yield. Nucleic acid amplification is typicallyachieved by the polymerase chain reaction (PCR). This method allows theamplification of specific, predefined nucleic acid sequences by the useof sequence-specific primer. Depending on the question to be solved, theamplified material might be further analyzed for example by sequencing.

With the progresses in the reliability and simplification of suchmethods, for example by the use of kits, these methods have becomestandard procedures in these different fields. Together with anincreasing demand for diagnostics based on molecular level, there is anincreasing need for the automated processing of relevant samples,starting with an initial biological sample through to the finalanalysis.

RELATED PRIOR ART

Automated liquid handling systems are generally well known in the art.An example is the Freedom EVO® robotic workstation from the presentapplicant (Tecan Schweiz AG, Seestrasse 103, CH-8708 Mannedorf,Switzerland). This device enables automated liquid handling in astand-alone instrument or in automated connection with an analyticalsystem. These automated systems typically require larger volumes ofliquids (microliter to milliliter) to process. They are also largersystems that are not designed to be portable.

A portable device for lysing and/or purifying biological samples isknown from WO 2007/061943. The processing of nucleic acids is performedwithin a cartridge chamber using electrodes arranged on the two sides,thus processing biological material by electrolysis, electroporation,electro-osmosis, electrical kinetic or resistive heating. The cartridgefurther comprises sieving matrixes or membranes. By the use of adequatebuffers and other reagents, in combination with the application of theelectrodes, various reactions can be performed within the chamber, anddesired products can be directed for example to collecting membranes.The cartridge itself can be placed into an integrated system comprisingthe required control elements and energy sources. Although thiscartridge provides a system to at least partially control the sampleprocessing electronically, intervention of an investigator or oftechnical lab staff is still required.

Other approaches to deal with the automated processing of biologicalsamples originate from the field of microfluidics. This technical fieldgenerally relates to the control and manipulation of liquids in a smallvolume, usually in the micro- or nanoscale format. Liquid movement in achannel system is known per se as, e.g. being controlled by micro pumpsin stationary devices or centripetal forces in rotating labware. Indigital microfluidics, a defined voltage is applied to electrodes of anelectrode array, so that individual droplets are addressed(electrowetting). For a general overview of the electrowetting method,please see Washizu, IEEE Transactions on Industry Applications, Volume34, No. 4, 1998, and Pollack et al., Lab chip, 2002, Volume 2, 96-101.Briefly, electrowetting refers to a method to move liquid droplets usingarrays of microelectrodes, preferably covered by a hydrophobic layer. Byapplying a defined voltage to electrodes of the electrode array, achange of the surface tension of the liquid droplet, which is present onthe addressed electrodes, is induced. This results in a remarkablechange of the contact angle of the droplet on the addressed electrode,hence in a movement of the droplet. For such electrowetting procedures,two principle ways to arrange the electrodes are known: using one singlesurface with an electrode array for inducing the movement of droplets oradding a second surface that is opposite a similar electrode array andthat provides at least one ground electrode. A major advantage of theelectrowetting technology is that only a small volume of liquid isrequired, e.g. a single droplet. Thus, liquid processing can be carriedout within considerably shorter time. Furthermore the control of theliquid movement can be completely under electronic control resulting inautomated processing of samples.

A device for liquid droplet manipulation by electrowetting using onesingle surface with an electrode array (a monoplanar arrangement ofelectrodes) is known from the U.S. Pat. No. 5,486,337. All electrodesare placed on a surface of a carrier substrate, lowered into thesubstrate, or covered by a non-wettable surface. A voltage source isconnected to the electrodes. The droplet is moved by applying a voltageto subsequent electrodes, thus guiding the movement of the liquiddroplet above the electrodes according to the sequence of voltageapplication to the electrodes.

An electrowetting device for microscale control of liquid dropletmovements, using and electrode array with an opposing surface with atleast one ground electrode of is known from U.S. Pat. No. 6,565,727 (abiplanar arrangement of electrodes). Each surface of this device maycomprise a plurality of electrodes. The drive electrodes of theelectrode array are preferably arranged in an interdigitatedrelationship with each other by projections located at the edges of eachsingle electrode. The two opposing arrays form a gap. The surfaces ofthe electrode arrays directed towards the gap are preferably covered byan electrically insulating, hydrophobic layer. The liquid droplet ispositioned in the gap and moved within a non-polar filler fluid byconsecutively applying a plurality of electric fields to a plurality ofelectrodes positioned on the opposite sites of the gap.

The use of such an electrowetting device for manipulating liquiddroplets in the context of the processing of biological samples is knownfrom the US patent application No. 2007/0217956 A1. Here it is suggestedto amplify nucleic acids on a printed circuit board for example throughthermocycling. The electrodes are transported on an array of electrodesby applying a potential between a reference electrode and one or moredrive electrodes. The sample is placed into a reservoir on the printedcircuit board, and droplets are dispensed on said printed circuit board.

However, none of the known devices allow the fully automated processingof nucleic acids starting from collected material up to the finalanalysis in the small volume scale. An additional disadvantage of thepresented devices comes with the nature of such arrangements ofelectrode arrays, being generally expensive in production, thus beingrather non-disposable in use. A continuous re-use of the same device fordifferent biological samples and applications however bears the risk ofcross-contaminating the samples of interest, which could lead to falseresults. Therefore, such devices are not suited for high-throughputassays.

OBJECTS AND SUMMARY OF THE PRESENT INVENTION

It is an object of the present invention to suggest a device whichenables the fully integrated handling of biological samples in a simple,automated and rapid manner, starting the handling with the provision ofa sample to be analyzed for its biological material into the device andfinalizing the processing with the achievement of a final analysis.

This object is achieved according to a first aspect by a biologicalsample processing system as herein described and disclosed.

It is a further object of the present invention to suggest analternative device which enables a simplified manipulation of liquiddroplets in a microscale format.

This object is achieved according to a second aspect by providing aliquid droplet manipulation system which enables a precise and guidedmovement of liquid droplets in a microscale format.

Additional preferred features according to the present invention resultfrom the dependent claims.

Advantages of the present invention comprise:

-   -   The system provides a multi-component device adjusted for a        fully automated processing of biological samples up to the        analysis.    -   Such a fully integrated system that can directly accept        macro-volumes of sample (either in liquid form or on a solid        surface such as a buccal swab) and process utilizing        nano-volumes; all without any user interaction.    -   The distinction between disposable and non-disposable components        allows the automated processing in a standardized and        cost-efficient manner.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be explained in more detail on the basis ofexemplary embodiments and schematic drawings. These explanations howevershould not restrict the scope of the present invention. Furthermore, therelative dimensions shown in the figures may vary considerably, as theseschemes are not drawn to scale. There is shown in:

FIG. 1 a schematic cross section and partial layout of a biologicalsample processing system according to the first aspect of the presentinvention;

FIG. 2 a top view on a container and a film of a biological sampleprocessing system according to the present invention, wherein

FIG. 2A shows the at least one well for positioning the biologicalsample being arranged towards the outer margin of the container, and

FIG. 2B shows the at least one well for positioning the biologicalsample being arranged in the center of the container;

FIG. 3 top views of different embodiments of preferred electrode arrays,wherein in

FIG. 3A each electrode is accomplished in form of a rectangle;

FIG. 3B each electrode is accomplished in form of a hexagon;

FIG. 3C each electrode is accomplished in form of a circle; and

FIG. 3D each electrode is accomplished in form of a triangle.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a schematic cross section and partial layout of anexemplified biological sample processing system 1 according to the firstaspect of the present invention. For enabling the processing ofbiological samples in an automated and cost-efficient manner, thissystem 1 comprises distinct single components which can be assembled toone unit, the system 1, in simple steps. Such component is for example acontainer 2 which is comprised by the biological sample processingsystem 1. The container 2 is accomplished for processing large volumesof liquid 18. In the context of the present invention, large volumes ofliquid are understood to relate to liquid volumes up to 5 ml or up to 10ml, depending on the sample to be hosted. For example, in case a buccalswab is used, the large volume well is preferably designed to holdvolumes up to 2 ml; if holding for example whole blood, the wellpreferably holds up to 5 ml. The container 2 has a top side 3 and a baseside 4. At its base side 4, the container 2 comprises protrusions 5.These protrusions 5 may be accomplished as parts of the container 2extending downwardly at the base side 4. Alternatively these protrusions5 may be attached to the base side 4 of the container 2 separately, forexample by gluing, welding or other appropriate means to stably attachsuch protrusions 5 to the base side 4 of the container 2. The container2 comprises at least one well 6. This at least one well 6 is open at itstop side 7. Thus, a biological sample 9, a reaction reagent 10, or bothcan be positioned within this well 6. At its bottom side 8, the at leastone well 6 has at least one opening 11. This opening is connected by achannel 12 of the container 2 with an orifice 13 of the container 2 atits base side 4. In the case a liquid 18 or a liquid droplet 19 isplaced into the at least one well 6 (with or without a reaction reagent,and/or with or without at least parts of the biological sample 9), itcan be transferred out of the well 6 through the channel 12. Thediameter of the channel 12 preferably is chosen such that the capillaryforce prevents liquid from leaking out the orifice 13 and that theliquids thus stay inside the at least one well 6 without need for avalve or any other closure of the channel 12. The diameter of thechannel 12 preferably is from 100 μm to 1 mm.

Furthermore the biological sample processing system 1 comprises a flatpolymer film 14. This flat polymer film 14 could also be called a“plastic skin” as proposed by Yang et al. (2008) “Exchangeable,pre-loaded “Skin Depot” for digital microfluidics” at the MicroTASmeeting in San Diego, Calif. This flat polymer film 14 preferably has alower surface 15 and a hydrophobic upper surface 16. As a material forthe thin polymer films, food wraps, and stretchable wax films can beused. When assembling the single components to the biological sampleprocessing system 1 in a first step by positioning the container 2 onthe film 14, the hydrophobic upper surface 16 of the film 14 is abuttedby the protrusions 5 of the container 2. Thereby, the protrusions 5 keepthe flat polymer film 14 at a distance “d” to the base side 4 of thecontainer 2. This distance “d” is set by the height of the protrusions 5of the container 2, and defines at least one gap 17 when the container 2is positioned on the flat polymer film 14. The gap 17 between the upperhydrophobic surface 16 of the film 14 and the base side 4 of thecontainer is sized to accommodate a liquid droplet. Preferably, this gap17 is less than 2 mm. Most preferably, the gap 17 is less than 1 mm.

The biological sample 9 preferably is contained a well 6. It can bemixed with a liquid 18, such as a buffer solution with or without lysisreagents. The biological sample 9 may be displaced from the at least onewell 6 (while kept within a liquid droplet 19), through the channel 12of the container 2 onto the hydrophobic upper surface 16 of the flatpolymer film 14. The liquid droplet 19 with the biological sample 9 isthus placed in the gap 17 between the film 14 and the container 2.

The displacement may be performed, without using valves, by a pressureapplied, centrifugal force, or electrowetting against the capillaryforces that prevent leaking of liquids out of the wells 6,6′. However,also other means may be used which are suitable to displace the liquid18 or liquid droplet 19 from the well 6 onto the hydrophobic uppersurface 16 of the flat film 14. These means for displacement may also beused for transferring a reaction reagent 10, which is stored in a well6′ of the container 2, onto the upper surface of the film. Whendisplacing liquids from the wells 6,6′ onto the upper polymer filmsurface 16, excess air form the gap may be vented e.g. via an empty well6′.

For the manipulation of liquid droplets 19, which had been preferablydisplaced from the at least one well 6 of the container 2 onto the uppersurface 16 of the film 14, the biological sample processing system 1comprises furthermore a liquid droplet manipulation instrument 20. Thisinstrument 20 comprises at least one electrode array 21, a substrate 22which supports the at least one electrode array 21 and a control unit23. The liquid droplet manipulation instrument 20 is accomplished sothat the container 2 and the film 14 can be reversibly attached to theinstrument 20. Thereby, the lower surface 15 of the film 14 abuts theelectrode array 21. When assembled in this a way, the biological sampleprocessing system 1 enables the displacement of a liquid droplet 19 fromthe at least one well 6 of the container 2 onto the upper surface 16 ofthe flat polymer film 14 and accordingly above the at least oneelectrode array 21. The electrode array 21 is accomplished to inducemovements of the liquid droplets 19. Thus, the instrument 20 isaccomplished to control a guided movement of said liquid droplet 19 onthe upper surface 16 of the flat polymer film 14 by electrowetting andto process there the biological sample 9.

Typical biological samples 9 which are processable by a biologicalsample processing system 1 are nucleic acids or proteins. Preferably,nucleic acids are used for processing. Such nucleic acids comprise DNA(desoxyribonucleic acid, for example genomic DNA, cDNA, mtDNA), RNA(ribonucleic acid, for example mRNA), being single- or double stranded,and derivatives thereof (for example artificially labeled nucleic acids.These biological samples 9 may be contained in tissue samples such asoral mucosa cells or hair follicles. Likewise the biological samples 9may be contained in a liquid, such as samples of body fluids such asblood, urine, sputum etc. A biological sample 9 of interest can beprocessed by a biological sample processing system 1 according to thepresent invention independent of its origin. Of particular interest aresamples taken for example from patients (in routine diagnosticprocedures) or from a crime scene (in criminal forensics). However, fora successful processing of the sample, the selection of requiredreaction reagents 10 should be adopted based on the material whichcomprises the biological sample 9. It is also possible to load analready purified biological sample 9 into the at least one well 6 of thecontainer 2. In this case, a purification step is not necessarilyrequired during the processing within the biological sample processingsystem 1.

Preferably, the at least one well 6 of the container 2 is sized toaccommodate a solid substrate 24 which carries the biological sample 9.This solid substrate 24 might be a tissue sample. However, it is alsopossible that this solid substrate 24 is a swab, a spatula, a needle, asyringe, a piece of paper such as FTA paper, or fabric material such asclothing, or other substrate suitable for carrying and/or collecting abiological sample 9 or e.g. tissues comprising the sample 9. Mostpreferably, the solid substrate 24 is a swab head, and accordingly, theat least one well 6 of the container 2 is sized to accommodate a swabhead. An exemplary embodiment of a well 6 accommodating a swab head isshown in FIG. 1. A typical size of such a well 6 for a swab head has adiameter of about 10 mm and a height of about 40 mm. Such a swab headmay be made for example of cotton or polyester, as commonly known in theart. These solid samples 24 may carry as well tissue samples orbiological samples 9 in form of a liquid (such as bodily fluids).

In the present FIG. 1, a container 2 is shown to comprise one well 6sized to accommodate a swab head, and further wells 6′ which aredifferent from the sample well 6 in size. In another variant, as shownin FIGS. 2A and 2B, the container 2 comprises at least one sample well 6and six smaller wells 6′ for storing reaction reagents. These wells 6′are preferably sized to store reaction reagents 10 and other requiredliquid such as buffers. A typical size of such a well 6′ for storingreaction reagents 10 has a diameter of about 5 mm and a height of about40 mm. However, the size of each well 6,6′ of the container 2 may beadopted individually according to the requirements given by theunderlying question to be solved. Similarly, the position of the wells6,6′ within the container 2 may be adopted as depending on the design ofthe liquid droplet manipulation instrument 20, production methods etc.Preferably the wells 6,6′ are positioned in the outer regions of thecontainer 2, to provide a central area beneath the container for movingliquid droplets 19 and for processing the sample 9 within the liquiddroplet 19. For processing, drops of reaction reagents 10 can betransferred onto the hydrophobic upper surface 16 of the flat polymerfilm 14 and mixed there with a droplet 19.

The at least one well 6 having a solid substrate 24 carrying thebiological sample 9 may also comprise a reaction reagent 10. Preferably,such a reaction reagent 10 is suited to free the biological sample 9from the material it is contained in. A lysis reagent for example wouldbe well suited for these purposes. It might comprise a reaction bufferand means to enzymatically open the cellular envelope hosting thebiological sample 9. The reaction reagent 10 may be positioned withinthe well 6 in form of a liquid. Depending on the application andavailability however, the reaction reagent 10 may alternatively bepositioned within the well 6 for example in a lyophilized form. Thisform of reaction reagent 10 is preferred in the case when the containershould be purchasable having preloaded reaction reagents 10. However itis the general knowledge of a skilled person that the lyophilized formof a reaction reagent is only preferred when the lyophilization processhas no or only minor influences on the functionality of the reagent 10.

In a preferred embodiment, the container 2 of the biological sampleprocessing system 1 comprises at least one well 6 which is accomplishedas storage for a reaction reagent 10. This embodiment is particularlypreferred in case the container 2 comprises already one well 6 forpositioning a biological sample 9. Thus, in this situation, thecontainer comprises at least two wells 6, one well 6 for positioning thebiological sample 9 and one well 6′ for storing a reaction reagent.Stored reagents 10 comprise reagents selected from a group comprisingreagents for performing cell lysis, reagents for performing nucleic acidpurification, reagents for performing nucleic amplification and reagentsfor performing sequencing of nucleic acids.

During cell lysis, the cellular integrity is disrupted by opening cellmembranes. This step can be performed using for example enzymaticactivity or chemical lysis. However other procedures to disrupt cellularintegrity may be suitable. Exemplarily, the thermostable protease EA1manufactured by ZyGem™ Corporation (Waikato Innovation Park, RuakuraRoad, Hamilton, New Zealand) should be mentioned at this place as asuitable enzyme for performing cell lysis. Alternatively, cell lysis maybe carried out using Proteinase K, or chemical lysis, both proceduresalso well known in the art. The buffer matching to the used enzyme canbe chosen by a skilled person without the need of special efforts and isregarded to be based on the general knowledge in the art, too. As theprocedure of performing cell lysis is well known to a skilled person, itshould not be described here in more detail.

DNA purification processes are well known in the art, too, and thesingle procedural steps should not be explained here. A purificationstep is preferred in such cases where the sample mixture compriseselements which might distract following reactions. In the context ofthis application, such a purification step is desired preferably after acell lysis or after nucleic acid amplification processes such aspolymerase chain reaction or sequencing by synthesis. Preferably, DNA isto be purified. Typically, reagents for performing nucleic acidpurification comprise beads or particles, eventually modified, which arecapable to bind DNA directly or indirectly. After DNA-binding, undesiredcontents of the sample mixture can be washed off, and DNA can beresolved in a desired liquid. Such beads may be standard magnetic beadswell known in the art. Advantageous beads include DNA IQ™ offered fromPromega Corporation (2800 Woods Hollow Road, Madison, Wis. 53711 USA) orDynal® Magnetic Beads offered from Invitrogen Ltd (EuropeanHeadquarters: 3 Fountain Drive, Inchinnan Business Park, Paisley PA49RF, UK). Suitable beads or particle may also be modified. Such amodification may simplify and specify the purification, as is mediatesbinding of specifically labeled DNA. DNA labeling can be achieved duringan amplification process. A typical label used for primer in polymerasechain reaction is biotin; however, other labels suitable can be used inthe context of the present invention. The labeled primer, incorporatedinto the amplicon, can be captured in the subsequent purificationprocess using for example streptavidin coated beads. However, othersystems suitable for the purification of amplified DNA may be used. Forexample Dynal® Magnetic beads may be used also in this secondpurification step.

Polymerase chain reaction (PCR) is typically used for the amplificationof nucleic acid and is also well known in the art. Shortly, PCRcomprises a cyclic repetition of three basic, temperature specificsteps: a nucleic acid denaturation step separating the double strands ofDNA at preferably 98° C., an annealing steps allowing preselected primer(oligonucleotides) to bind to respective sequences on the single strand,wherein this temperature step depends on the primer sequence, and anextension step involving a polymerase which extends bound primer to anucleic acid strand at an enzyme specific temperature. The polymerase ispreferably thermostable, so that it is not influenced by thedenaturation temperature. Such a thermostable polymerase well known inthe art is the polymerase of the bacterium Thermus aquaticus(Taq-polymerase). However, other thermostable polymerases available maybe used. Preferred templates are genomic DNA or cDNA. Using PCR,pre-selected, specific regions of a template may be amplified, givingfor example more information about the origin of the DNA. Preferredregions to be analyzed by PCR comprise mitochondrial DNA (mtDNA),typical short tandem repeats (STR), or distinct single nucleotidepolymorphisms (SNPs) known for being for example linked with specificdiseases (used as genetic markers).

Sequencing of specifically amplified DNA is a well-known tool to furthercharacterize the selected DNA. Major sequencing principles are known inthe art, sequencing by amplification and sequencing by hybridization.Sequencing by amplification involves a PCR-related process using howeverlabeled stop-primer which terminate the extension process randomly. Theresulting end-labeled fragments are then used for determining thesequence of the template. Sequencing by hybridization (SBH) involves thelinkage of labeled primer to a matrix. Primer are selected so that theoverlap partially in their sequence. After hybridization of a target DNAto said primer, sequence can be determined by the analysis of primersequenced to which the target is bound. When applying sequencing byhybridization step with the biological sample processing system 1, thelabeled primers are preferably linked to the hydrophobic upper surface16 of the flat polymer film 14 prior to the start of sample processing.Most preferably, the labeled primers are linked prior to the release ofthe system into the trade.

In the case, two or more, preferably all methods presented above shouldbe performed using the biological sample processing system 1 accordingto the resent invention, it is required that the container 2 comprisesmore than two wells 6,6′. Preferably, the container 2 comprises at leastone well 6 for positioning a biological sample 9 and further wells 6′for storing the required reaction reagents 10, with one dedicated well6′ for each reaction reagent 10 of one method. Preferably, the well 6for positioning the biological sample 9 is accomplished to storeadditionally reaction reagents 10 and buffers required for cell lysis.Cell lysis can thus be performed directly in the well 6 which holds thebiological sample 9.

Should all methods mentioned above be performed, the container 2 thencomprises at least four wells 6,6′: one well 6 for positioning thebiological sample 9 and storing reaction reactions 10 for cell lysis,one well 6′ for storing reaction reagents 10 for DNA purification, onewell 6′ for storing reaction reagents for PCR, and one well 6′ forstoring reaction reagents 10 for sequencing. Most preferably, thecontainer 2 comprises at least ten wells 6,6′ for processing of abiological sample 9:

-   -   at least one well 6 is accomplished for positioning the        biological sample 9, for storing reaction reagents 10 and buffer        and for performing cell lysis,    -   at least three wells 6′ are accomplished for pre-PCR        purification (one each for magnetic beads, wash buffer, and        elute buffer),    -   at least two wells 6′ are accomplished for amplification (one        for storing the enzyme and buffer, one for storing the primer,        with one primer well per locus to be amplified),    -   at least two wells 6′ are accomplished for post-PCR clean-up        (one for storing streptavidin coated beads and one for storing        wash buffer),    -   at least two wells 6′ are accomplished for storing reaction        reagents 10 and buffer for sequencing by hybridization (one for        storing buffer comprising a reference probe and one for storing        buffer comprising a probe of interest).

Generally, the number of wells 6,6′ is dependent on the type of reactionsystem used (required reagents, processing steps) and the number ofanalysis required (number of sequences/loci to be analyzed, i.e. STR,SNP, mtDNA) and may be adopted by a skilled person based on generalknowledge in the art. If the primer for the amplification should bestored in the container 2, preferably the container 2 comprises oneprimer well per loci to be analyzed for the amplification process. Thus,in case 16 loci should be analyzed, the container preferably comprises16 primer wells 6′ for the amplification. In another preferred variant,the primer required for the amplification step may be available in driedform on the hydrophobic upper surface 16 of the flat polymer film 14, sothat for storing primer, no separate well 6′ would be required here inthe container 2. The primer may in this case be re-suspended on the film14 using a buffer held in a well 6′. For the sequencing by hybridizationstep, the number of wells 6′ required to store reaction reagents 10 andbuffer may similarly be adopted.

In an especially preferred embodiment, the biological sample processingsystem 1 is accomplished to perform the extraction, purification,amplification and analysis of a biological sample 9 of interest. Thus,the present invention provides according to the first aspect a fullyintegrated system that can directly accept macro-volumes of sample(either in liquid form or on a solid surface such as a buccal swab) andprocess utilizing nano-volumes up to the final analysis.

In the FIGS. 2A and 2B, a container 2 according to the present inventionis shown in a top view, having one well 6 for positioning the biologicalsample 9 and six further wells 6′ for storing reaction reagents 10.

When cell lysis is performed directly within the well 6 for positioningthe biological sample 9, the biological sample 9 is set free from thecellular context, and preferably released into a liquid 18, whichtherefore is a reaction solution resulting from the cell lysis. In casethe biological sample 9 was not contained in one or more cells whenpositioned into the well 6 of the container 2 (if lysis is notrequired), the liquid can be chosen according to the followingprocedural steps, and added into the well via the top side 7 of the well6. In each case, the biological sample 9 should be contained at least inparts in a liquid for the further processing using the biological sampleprocessing system 1 according to the first aspect of the presentinvention. The liquid 18 or a liquid droplet containing at least partsof the biological sample 9 is then displaced for further processing fromthe well 6 through the channel 12 of the container 2 onto thehydrophobic upper surface 16 of the flat polymer film 14.

The displacement preferably performed, without using valves or othermoveable means, by a pressure applied, by centrifugal force, or byelectrowetting. All these preferred displacement means act against thecapillary forces that prevent leaking of liquids out of the wells 6,6′.However, also other means may be used which are suitable to displace theliquid 18 or liquid droplet 19 from the well 6 onto the hydrophobicupper surface 16 of the flat film 14.

As for further processing, the container 2 and the flat polymer film 14are reversibly attached to the liquid droplet manipulation instrument20, with the lower surface 15 of the film 14 abutting the electrodearray 21. Accordingly, the liquid droplet 19 is displaced from the well6 above the electrode array 21. In this arrangement, the liquid droplet19 may be moved in a guided manner by the liquid manipulation instrument20 by electrowetting. The movement is controlled to achieve the selectedprocessing of the biological sample 9 contained within said liquiddroplet 19 and to carry out this processing at preferred sites of theelectrode array.

In a variant of the biological sample processing system 1, the liquiddroplet 19 is moved in the gap 17 within an immiscible system liquid 32.This variant is the preferred embodiment, when performing PCR on thebiological sample 9 contained in the at least one liquid droplet 19. AsPCR requires the exposure of such a liquid droplet 19 to differenttemperatures, including the denaturation step at about 98° C.,evaporation of liquid may be prevented or at least considerably reducedwith the use of such an immiscible system liquid 32. Preferred systemliquids immiscible with the liquid droplet 19 are selected e.g. fromsilicon oil, hexadecane and benzene.

For a form-fitted attachment of the container 2 to the liquid dropletmanipulation instrument 20, both, container 2 and instrument 20preferably comprise each at least one positioning element 25. Suchpositioning elements are preferably selected from a group comprising:

-   -   at least one groove in the lateral area 28 of the container 2        and at least one elevation extending from the instrument 20 in        such a way that when the container 2 with the film 14 is        attached to the instrument 20, groove and elevation are arranged        form-fitting to each other;    -   at least one groove on the base side 4 of the container 2 and at        least one elevation extending from the instrument 20 in such a        way that when the container 2 with the film 14 are attached to        the instrument 20, groove and elevation are arranged        form-fitting to each other;    -   at least one groove on the base side 4 of the container 2 and at        least one elevation extending from the instrument 20 in such a        way that when the container 2 with the film 14 are attached to        the instrument 20, groove and elevation are arranged        form-fitting to each other, wherein the at least one elevation        extending form the instrument 20 is a Peltier element for        locally providing the container 2 with a preselected        temperature; and    -   the container 2 having an irregular polyhedron shape and the        liquid manipulation instrument having a corresponding groove, so        that when attaching the container 2 to the instrument 20, both        are aligned in a form-fitted snugly manner.

Positioning elements 25 accomplished as at least one groove of thecontainer 2 and at least one elevation extending from the instrument 20are presented in FIG. 1 (with the groove at the base side 4 of thecontainer 2), FIG. 2A (with two triangular-shaped grooves in the lateralarea of the container) and in FIG. 2B (with two semi-circular shapedgrooves in the lateral area of the container 2). When using a Peltierelement for heating the well 6 for positioning the biological sample 9,such Peltier element can be accomplished as an elevation extending fromthe instrument 20, its position may be chosen so that for example, isspecifically provides the well 6 (whether this is central or not) with adefined temperature. However, other means for positioning the container2 on the liquid droplet manipulation instrument 20 in a definedconfiguration may as well be used which are well known to a skilledperson, and should not be described in more detail here.

While the container 2 is positioned on the liquid droplet manipulationinstrument 20, a liquid droplet 19 on the flat polymer film 14 mayeither contact only the hydrophobic upper surface 16 of the film 14 ormay contact both, the hydrophobic upper surface 16 of the film 14 andthe base side 4 of the container 2. The contact surfaces of such aliquid droplet 19 may be influenced by the sizing of the gap 17 (thus,sizing the protrusions 5) or by sizing the liquid droplet 19.

The container 2 is preferably made by injection molding. In this way,the production costs may be reduced despite the achievable highmanufacturing quality and the container 2 can be utilized as a low costdisposable. Such a single-serving container 2 is suited to be sold forvarious applications and can be equipped with a specific set of reactionreagents 10. The container 2 is preferably made either of anelectrically insulating material 26, of an electrically conductivematerial 27, or of a combination of both an electrically conductive andan electrically insulating material 26,27. When made of two differentmaterials, a two-step injection process is preferred. In the FIGS. 1,2A, and 2B, the core of the container 2 is made of an electricallyinsulating material 26, wherein the regions surrounding the wells 6,6′are made from an electrically conductive material 27. The surroundingregions made of the conductive material 27 are separated from each otherby the insulating material 26. These surrounding regions made ofconductive material 27 may form at the base side 4 of the container 2 anozzle 47, which slightly extends into the gap 17 (see FIG. 1). Theadvantage provided by such a nozzle is the possibility to distinctlyproduce and deliver a liquid droplet 19 into the gap without the droplet19 contacting the surface of the base side 4 of the container 2.Furthermore such a nozzle may enable a directed delivery of the droplet19 into the gap 17.

Furthermore, parts of the surrounding regions made from the electricallyconductive material 27 form a part of the outer, lateral side 28 of thecontainer 2. Such a variant has the advantage, that each electricallyconductive region 27 of the container 2 may be individually contactedelectrically. This allows the conductive regions to be addressed by avoltage control 29 and provided with an individual voltage. Thus, fromeach well 6,6′ one or more liquid drops may be displaced to thehydrophobic upper surface 16 of the flat polymer film 14 using theprinciple of electrowetting. Importantly, the displacement can be donefor each well individually, so that reaction reagents 10 or liquidscontaining the biological sample 9 may be individually displaced at thetime they are required on the film 14.

In FIG. 2A, the at least one well 6 for positioning a biological sample9 is arranged towards the outer, lateral side 28 of the container 2. Theat least one well 6 is additionally surrounded from an electricallyconductive material 27. The electrically conductive surrounding 27 is inthis variant extends to form the major part of the core of the container2. In this way, major parts of the base side 4 of the container 2 aremade of electrically conductive material 27, too. This allows processinga liquid droplet 19, which is positioned on the hydrophobic uppersurface 16 of the film 14 and which contacts the base side 4 of thecontainer 2, by electrowetting using the electrically conductive partsof the base side 4 of the container 2 as a ground electrode.Accordingly, in this variant, the guided movement of the liquid droplet19 may be further stabilized.

When the container 2 itself is subjected to a heating step, for exampleto promote cell lysis within the at least one well 6 for positioning thebiological sample and/or a reaction reagent, it is preferred that partof the container 2 is made from a thermally isolating material or thatthermally insulating gaps are provided around the zone of highertemperature.

In a preferred embodiment, the container 2 comprises means foridentification 30, which are selected from a group comprising a barcodeand an RFID (radiofrequency identification) tag or another integratedchip. As such means for identification 30 are well known to the personskilled in the art, they should not be described in more detail here.Identification means 30 are especially preferred when the container 2 ofthe biological sample processing system 1 is used in an automatedmanner, while storing information for example about the biologicalsample positioned in a well 6 of the container 2. In addition, trackingof a particular sample is possible even in a large laboratory system.

When a solid substrate 24 comprising the biological sample 9 ispositioned in the at least one well 6 of the container, this well 6preferably comprises retention means 31 for preventing the solidsubstrate 24 to block the opening 8 of said well 6. The retention means31 are selected from a group comprising a filter, a frit (see FIG. 1)and relief structures (see FIG. 2B). However, other retention means 31well known in the art may be used for these purposes.

The FIGS. 2A and 2B each show a container 2 having an analyzing area 33.A container 2 having an analyzing area 33 is preferred, when certainareas of the hydrophobic upper surface 16 of the flat polymer film 14should be accessible by optical means 38. In the simplest embodiment, acut-out section defines the analyzing area 33, preferably in the outerlateral side 28 of the container 2. The respective region of thehydrophobic upper surface 16 of the film 14, underlying the cut-out, isin this way accessible for optical means 38. Such optical means are e.g.a human eye or an optical device. FIGS. 2A and 2B show exemplarily apreferred position of an optical means in relation to the analyzing area33. Most preferably, the analyzing area 33 is positioned above thatregion of the hydrophobic upper surface 16, which is accomplished to theprocessing of a biological sample 9 using sequencing, especiallypreferred when as the sequencing method sequencing by hybridizationmethod is performed. Preferably, optical devices 38 are selected from agroup comprising a standard microscope, a camera system, a light guidingsystem such as fiber optics, a scanner, and adaptations or combinationsthereof. For example, in a very simple embodiment, a camera, a simpleCCD or a PMT (Photo Multiplier Tube) is used together with a lightsource, such as an LED, which serves as an excitation source forfluorescent tags on the film 14. If a light guiding system is used, theexcitation- and/or measurement device may be located aside of thecontainer 2. Thus, automatic sample processing and final analysis can becarried out on the same polymer film 16 and on the electrode array 21.

As shown in the FIGS. 2A and 2B, the container 2 preferably comprises asupport rim 45 when having an analyzing area 33. This support rim 45extends along the outer lateral side 28 of the container 2 whilebordering the analyzing area 33. Accordingly, this support rim 45 mayalso comprise one or more protrusions 5 at its bottom side, which areattached to the hydrophobic upper surface 16 of the flat polymer film14. This support rim 45 supports the container 2 having a cut-out whenpositioned on the film 14.

In a special user friendly variant, a multitude of containers 2 havingat least one analyzing area 33 is arranged in such a way, that eachanalyzing area 33 is easily accessible by one optical device 38. Onepossible way would be an essentially circular arrangement of thecontainers 2 around a rotary optical device 38. Alternatively, thecontainers 2 can be stored in vertical or horizontal rows of adequatecarrier, and the optical device 38 or the carrier with the row ofcontainer 2 are shifted manually or automatically into a position inwhich the analyzing 33 is accessible by the optical device 33.

Both, the container 2 and the flat polymer film 14 can be provided tothe user either as separate components that remain to be assembled whenthe processing of a biological sample 9 is to be started. In analternative embodiment however, these two components can be provided asa cartridge 40. In this case, the cartridge comprises both, container 2and the flat polymer film 14, which are attached to one another forexample by gluing or welding or other appropriate means to stably attachthese two components.

Preferably, the container 2 or the cartridge 40 comprises a cover 43 forprotecting the wells 6,6′ and their content from outside influences.Such a cover 43 may be sealingly attached to the top side 7 of thecontainer 2. The attachment may be reversible. In a preferred variant,the cover 43 is a thin film, which optionally is made of a pierceablematerial. In this way, the wells 6,6′ of the container 2 may bepreloaded. Safe storage is allowed by applying the film cover 43 ontothe container 2. Only upon start of the sample processing, the filmcover 43 is pierced open and the wells 6,6′ of the container 2 areaccessible for the user. Additionally, the container 2 or the cartridge40 can be covered with a cover 43 as well.

In a second aspect, the present invention relates to a liquid dropletmanipulation instrument 20. In a preferred embodiment, this liquiddroplet manipulation instrument 20 is accomplished to be used in thebiological sample processing system 1 according to the first aspect ofthe present invention. However, the liquid droplet manipulationinstrument 20 may be used independently of the biological sampleprocessing system 1.

The liquid droplet manipulation instrument 20 according to the secondaspect of the present invention comprises at least one array ofelectrodes 21 for inducing a movement of a liquid droplet byelectrowetting. The liquid droplet manipulation instrument 20 alsocomprises a substrate 22 for supporting the electrode array 21, and acontrol unit 23. The control unit 23 comprises at least one electrodeselector 34, which is connected with at least one voltage control 29.The electrode selector 34 is accomplished to individually select eachelectrode 35 of the electrode array 21. Furthermore, the electrodeselector 34 is accomplished to provide the selected electrode 35 with avoltage which is controlled by the voltage control 29. At least theelectrode selector 34 and the voltage control 29 are controlled by acentral processing unit 36, which is comprised by the control unit 23.The central processing unit 36 is accomplished to control the electrodeselector 34 and the voltage control 29 to individually select at leastone electrode 35 and to provide the selected electrode 35 with anindividual voltage pulse. Preferably, the individual voltage pulse isselected from a group comprising a ground voltage and a drive voltage.With the selection and provision of an individual voltage pulse, theselected electrode 35 is defined as a drive electrode 35′ or as a groundelectrode 35″.

The electrodes 35 of the electrode array 21 may have various shapes.Generally, those shapes of electrodes 35 are preferred which aresuitable to establish an array of these electrodes 35. The FIGS. 3A-3Dshow some examples of preferred electrode-shapes. As can be seen in FIG.3A, the electrodes 35 may have a rectangular shape. Here, the electrodes35 have a square shape; however, other rectangular shapes may as well besuitable. In FIG. 3B, the electrodes 35 are shown as having a hexagonalshape, in FIG. 3C as having a circular shape and in FIG. 3D as having atriangular shape. However, other shapes may be suitable as well, as longas the electrodes 35 are able to establish an array and are accessibleby electrode contacting lines.

Preferably, the central processing unit 36 comprises activatablesoftware 37. This software 27 enables the central processing unit 36 tocontrol the electrode selector 34 and the voltage control 29 toindividually select at least one electrode 35 and to provide theselected electrode 35 with an individual voltage pulse.

The control unit 23 preferably comprises a power supply 44. This powersupply 44 provides at least the central processing unit 36 and thevoltage control 29 with electric power. Depending on the embodiments ofother elements, such as the electrode selector 34, the power supply 44may additionally provide also other elements with electric power.

The control unit 23 is capable to define a path for a guided movement ofa liquid droplet 19 by the selection of a series of subsequent driveelectrodes 35′. Thereby at least one of these selected drive electrodes35′ is subsequently provided with a drive voltage pulse along said path,under the control of the control unit 23. Furthermore the control unit23 is accomplished to essentially simultaneously provide at least oneelectrode 35″, which is adjacent to the pulsed drive electrode 35′ anddifferent to the selected drive electrode 35′ of the path, with a groundvoltage pulse.

Such a path for a guided movement of the liquid droplet is each shown inthe FIGS. 3A to 3D. The subsequent selected drive electrodes 35′ areindicated. The actual drive electrode 35′ is shown to be that electrode35, upon which the liquid droplet 19 is positioned. The direction of theplanned guided movement of the liquid droplet 19 is indicated with anarrow. In that direction, subsequent electrodes 35′ along the path willbe provided with a drive voltage pulse.

Preferably, the size, respectively the diameter of the liquid droplet 19slightly exceeds the diameter of an electrode 35. Most preferably, forthe guided movement by electrowetting, the liquid droplet touches notonly the actual drive electrode 35′ but slightly touches simultaneouslythe subsequent electrode 35′ which will become the next actual driveelectrode 35′. However, the adjustment of electrode size in relation toliquid droplet sizes is within the knowledge of the person skilled inthe art and should not be repeated here. However, the actual size anddesign of the electrodes and the desired size of the liquid droplets 19must be in accordance with each other and with the praxis ofelectrowetting.

According the second inventive aspect, the presence of at least oneground electrode 35″ adjacent to the liquid droplet 19 to be movedprovides a stabilizing effect to its movement. The FIGS. 3A-3D indicatethose electrodes 35″ that might be provided with a ground voltage pulse.Those ground electrodes 35″ are preferably adjacent to the pulsed driveelectrodes 35′ and different to the selected drive electrodes 35′ of thepath. The provision with the ground voltage pulse is preferably carriedout essentially simultaneously to the provision with the drive voltagepulse. Alternatively, the provision with the ground voltage pulse is tobe carried out simultaneously to the provision with the drive voltagepulse.

According to a preferred variant of the liquid droplet manipulationinstrument 20, the control unit 20 is accomplished to provide at leasttwo electrodes 35″ which are adjacent to the pulsed drive electrode 35′and different to the selected drive electrode 35′ of the path with aground voltage pulse. Preferably, these at least two selected groundelectrodes 35″ are subsequent electrodes 35 on the same side of thepath.

As shown in the FIGS. 3A-3B, ground electrodes 35″ may be selected fromelectrodes 35 along the path, adjacent to the path and adjacent to theliquid droplet 19. Preferably, the selected ground electrodes 35″ are onthe same side of the path. When a group of three or more electrodes 35″is to be provided with a ground voltage potential essentiallysimultaneously, at least two first electrodes 35″ are preferablyselected from one side of the path. The remaining electrodes 35″ of thatgroup may however be selected from that side of the path being oppositeto the first two ground electrodes 35″ of that group. However, even whena group of ground electrodes 35″ are selected from two sides of thepath, they are provided with the ground voltage pulse essentiallysimultaneously or simultaneously to the pulsed drive electrode 35′. If agroup of electrodes 35″ is simultaneously provided with a ground voltagepulse, the other electrodes 35″ may be adjacent to the path and ahead ofthe liquid droplet 19, adjacent to the path and behind the liquiddroplet 19 or both.

In one variant of the liquid droplet manipulation instrument 20, a groupof 2 or more electrodes 35 may be provided with a drive voltage pulseessentially simultaneously. In this case, a liquid droplet 19 of alarger volume may be moved. However in this variant it is preferred thatessentially simultaneously or simultaneously a group of 2 or moreelectrodes 35 are provided with a ground voltage pulse to sufficientlysupport the liquid droplet 19 with the larger volume.

In a preferred variant of the liquid droplet manipulation instrument 20,the control unit is accomplished to provide at least one selectedelectrode with a stop voltage pulse for generating a stop electrode35′″. Preferably, the provided stop voltage pulse is different to thedrive voltage pulse and the ground voltage pulse.

The voltage pulses for defining a selected electrode 35 as a driveelectrode preferably are between 20 and 100 V. The voltage pulses fordefining a selected electrode 35 as a stop electrode preferably arebetween −50 V and +50 V. As shown in the FIGS. 3A-3D, selected stopelectrodes 35′″ are adjacent to the path, different to the selecteddrive electrode 35′ of the path and different to the at least oneselected ground electrode 35″ adjacent to the path. Furthermore, stopelectrodes 35′″ are selected from such electrodes 35 adjacent to thepath, where the path provides a change of direction for the liquiddroplet 19 movement. A stop electrode 35′″ supports the direction changeof the liquid droplet movement along the path.

The FIGS. 3A-3D show exemplarily possible positions of stop electrodes35′″ along the path. Preferably, at least one electrode 35′″ along thepath at a place of direction change is provided with a stop voltagepulse. However, more than one electrode 35′″ in that area may beselected to be provided with a stop voltage pulse, as shown in FIG. 3D.Here, two or more electrodes are selected as stop electrode 35′″ at thepoint of direction change to support the liquid droplet movement.

The FIGS. 3B and 3C show exemplary a virtual grid of the electrode array21. Each grid point 39 of the virtual grid is established by thegeometrical center of each electrode 35 of the electrode array 21. FIG.3B shows a hexagonal grid according to the hexagonal shape and densepacking of each electrode 35 of the electrode array 21. FIG. 3C shows anorthogonal grid based on the orthogonal arrangement of electrode 35,this time exhibiting essentially circular shape. Preferably, subsequentelectrodes for defining the path, subsequent selected ground electrodes35′″, and/or subsequent selected stop electrodes 35′″ are defined by theclosest distance between two grid points 39 of that virtual grid. Inthis way a continuous liquid droplet movement may be ensured. Thehexagonal arrangement of the electrodes array 21 is preferred over theorthogonal arrangement because of the higher degree of freedom.

FIG. 1 shows exemplarily the position of the electrodes 35 in relationto the substrate 22. Preferably, the electrodes 35 of the electrodearray 21 are positioned in relation to the substrate 22, so that theupper surface of the electrodes 35 are aligned substantially flush withthe upper surface of the substrate 22. Alternatively, the electrodes 35of the electrode array are positioned within the substrate 22 andenclosed by it (see left hand side on FIG. 1). It is preferred toposition the electrodes 35 as close to the liquid droplets 19 aspossible in order to be able to reduce the voltage necessary forelectrowetting. Thus, electrodes 35 flush with the upper surface of thesubstrate 22 and very thin polymer films are particularly preferred. Asa material for the thin polymer films, e.g. food wraps, and stretchablewax films can be used.

In a preferred embodiment, the liquid droplet manipulation instrument 20according to the present invention is accomplished to accommodate acontainer 2 for large volume processing and to simultaneouslyaccommodate a flat polymer film 14 with a hydrophobic upper surface 16.In case a container 2 for large volume processing and a flat polymerfilm 14 comprising a hydrophobic upper surface 16 are attached to theliquid droplet manipulation instrument 20, a system is formed suited forbiological sample processing of a sample 9 positioned within thecontainer 2. Such a system preferably corresponds to the biologicalsample processing system 1 according to the first aspect of the presentinvention.

In another variant, the liquid droplet manipulation instrument 20according to the present invention is accomplished to accommodate acartridge 40. Said cartridge comprises a container 2 and a flat polymerfilm 14 as previously described herein. Container 2 and film 14 of thecartridge 40 are attached to one another by gluing or welding, or byother appropriate means to stably connect the container 2 and the film14. In case such a cartridge 40 is attached to this variant of theliquid droplet manipulation instrument 20, a biological sampleprocessing system 1 according to the first aspect of the presentinvention may be formed.

In one variant, the liquid droplet manipulation instrument 20 comprisesat least two or more electrode arrays 21. Preferably, the electrodearrays 21 are arranged essentially horizontal within the liquid dropletmanipulation system 20. In this variant, the instrument 20 isaccomplished to accommodate at least two or more containers 2 togetherwith two or more flat polymer films 14, or to accommodate at least twoor more cartridges 40. Preferably, the container 2 and the film 14 orthe cartridge 40 may be positioned essentially above the electrodearray. However, it is also possible to position these componentsessentially sideways or laterally, when the electrode arrays are notaligned essentially horizontally but essentially vertical.

In an especially preferred embodiment, the biological sample processingsystem 1 according to the first aspect of the present inventioncomprises a liquid droplet manipulation instrument 20 according to thesecond aspect of the present invention and as discussed in detail above.The embodiment of the liquid droplet manipulation instrument 20 as wellas the embodiment of the biological sample processing system 1 may bechosen by selecting the various features discussed above, depending onthe question addressed. If not stated otherwise, the various featurespresented within this application may all be combined with each other.

In FIG. 1, a biological sample processing system 1 comprising a liquiddroplet manipulation instrument 20 is shown. The liquid dropletmanipulation instrument 20 comprises a reception element 46 to safelyreceive the container 2 and the film 14. In the embodiment shown, thepositioning elements 25 of the liquid droplet manipulation instrument 20are comprised by the reception element 46.

FIG. 3A shows an electrode array 21 according to such an especiallypreferred embodiment of a biological sample processing system 1. ThisFigure shows an enlarged top view of a distinct section of FIG. 1,indicated as a rectangle with a dotted line. The position of the atleast one well 6 for positioning a biological sample 9 in relation tothe defined path on the electrode array 21 is indicated by a dottedcircular line in FIG. 3A. The opening 11 at the bottom of the at leastone well 6, the passage of the channel 12 or the orifice 13 at the baseside 4 of the container 2 respectively are indicated as an inner circleof a dotted line. A liquid 18 or a liquid droplet 19 is transferred fromthe well 6 through the channel 12 on the hydrophobic upper surface 16 ofthe flat polymer film 14, namely above the electrode array 21 of theliquid droplet manipulation instrument 20. A liquid portion is indicatedin the center of the electrode array 21 shown. This liquid portioncovers at least one selected drive electrode 35′ from the electrodepath. Preferably, the liquid portion covers at least partiallysubsequent electrodes 35′ from the path. According to FIG. 3A, theliquid portion covers additionally electrodes 35″ selected to beprovided with a ground voltage pulse. A liquid droplet 19 is separatedfrom the liquid portion by the provision of a drive voltage pulse to anelectrode 35′ subsequent to the initial drive electrode 35′ along thepath. The liquid droplet 19 is then guided on along the path in a firstdirection, and after a direction change, in a second direction. At theposition of the direction change, a stop electrode 35′″ is generated tostabilize the direction change.

After e.g. a lysis step performed in a well 6 of the container anddisplacing a liquid portion, preferably comprising at least parts of thebiological sample 9 of the well 6, further processing may be performedon the hydrophobic upper surface 16 of the flat polymer film 14. Forprocessing a liquid droplet 19 with a DNA purification step, the use ofmagnetic beads is especially preferred. In this case, the liquid dropletmanipulation instrument 20 of the biological sample processing system 1comprises preferably at least one magnet 41. This magnet 41 controls themagnetic beads during the processing in the gap 17 on the upperhydrophobic side 16 of the flat polymer film 14. Suitable magnets may beelectromagnets or permanent magnets. The magnet 41 is arrangedpreferably on that side of the substrate 22 of the instrument, which isnot covered by an electrode array 21. The magnet 41 is alternativelyarranged preferably on that side of the substrate 22 of the instrument20, which is not abutted by the flat polymer film 14.

For processing a liquid droplet 19, which preferably comprises abiological sample 9, with a heat dependent processing step such as aPCR, the liquid droplet manipulation instrument 20 of the biologicalsample processing system 1 preferably comprises at least one heatingelement 42. This heating element 42 is preferably arranged on that sideof the substrate 22 of the instrument 20, which is opposite to the sideof the substrate 22 being abutted with the flat polymer film 14. The atleast one heating element 42 is accomplished to provide at least onetemperature zone with a predefined temperature on the upper hydrophobicsurface 16 of the flat polymer film 14. If the liquid dropletmanipulation device 20 comprises one heating element 42, PCR may beperformed by keeping the liquid droplet 19 comprising a biologicalsample 9 within the single temperature zone, while changing thetemperature within that single zone accordingly. If two heating elements42 are used, a PCR may be done by moving the liquid droplet 19comprising a biological sample 9 between the two zones, wherein thetemperature of each zone is adopted according to the temperaturerequired for the cycling steps. When processing a liquid droplet 19comprising a biological sample 9 by PCR, the biological sampleprocessing system 1 comprises in an especially preferred variant atleast three heating elements 42 for providing at least three differenttemperature zones on the upper hydrophobic surface 16 of the flatpolymer film 14. FIG. 1 shows a biological sample processing system 1having three heating elements 42 underneath the support substrate 22 andopposite to the side being abutted with the flat polymer film 14. Eachtemperature zone has a predefined temperature to enable a PCR beingperformed on the upper hydrophobic surface 16 of the flat polymer film14. Most preferably, one temperature zone comprises a temperature fordenaturizing double stranded nucleic acid, one temperature zonecomprises a temperature enabling the annealing of pre-selected primer,and one temperature zone comprises a temperature enabling a polymeraseto elongate the annealed primer to the full strand. Additionally, thebiological sample processing system 1 may comprise a fourth heatingelement 42 providing a temperature of about 4° C. Utilizing at leastthree heating elements 42 underneath the support substrate 22 has theadvantage that the selected temperatures can be kept constantly over theentire reaction time and the droplets can be moved from one temperatureregion to another. This movement allows for rapid temperature changeswithin the droplets 19, which are much faster than achievable bychanging the temperature of the heater element 42 while keeping thedroplet 19 in place.

In a further variant of the biological sample processing system 1, alayer of low vapor pressure liquid connects the lower surface 15 of theflat polymer film 14 with the upper surface of the at least oneelectrode array 21 to reduce the formation of air bubbles in-between.Preferably, the low vapor pressure liquid is silicon oil; however, otherlow vapor pressure liquids may be used as well.

Similar reference numbers refer to similar parts, if they are notparticularly discussed in detail.

List of reference numbers:  1 biological sample processing system  2container  3 top side of the container  4 base side of the container  5protrusions of the container  6, 6′ well of the container  7 top side ofthe well  8 opening at the bottom side of the well  9 biological sample10 reaction reagent 11 opening of the well 12 channel of the container13 orifice of the container 14 flat polymer film 15 lower surface of thefilm 16 hydrophobic upper surface of the film 17 gap 18 liquid 19 liquiddroplet 20 liquid droplet manipulation instrument 21 electrode array 22substrate of the instrument 23 control unit 24 solid substratecomprising the biological sample 25 positioning element 26 electricallyinsulating material 27 electrically conductive material 28 outer lateralside of container 29 voltage control 30 means for identification 31retention means 32 system liquid 33 analyzing area 34 electrode selector35 electrodes 35′ drive electrode 35″ ground electrode 35′″ stopelectrode 36 central processing unit 37 software 38 optical means 39grid point 40 cartridge 41 magnet 42 heating element 43 cover 44 powersupply 45 support rim 46 reception element 47 nozzle d distance betweenthe upper surface of the film and the base side of the container

What is claimed is:
 1. A cartridge (40) for enabling droplet manipulation by electrowetting and processing of biological samples in a liquid droplet manipulation instrument (20), wherein the cartridge (40) comprises: a container (2) having a top side (3) and a base side (4) and comprising at least one well (6) open at the top side (3), wherein the at least one well (6) comprises a bottom side (8) having at least one opening (11), the container (2) further comprising a channel (12) connecting the opening of the well (11) with an orifice (13) on the base side (4) of the container (2), and wherein the base side (4) of the container (2) comprises protrusions (5) distributed thereon; and a flat polymer film (14) having a lower surface (15) and a hydrophobic upper surface (16), which is kept at a distance (d) to the base side (4) of the container (2) by the protrusions (5), the distance (d) defining at least one gap (17) when the container (2) is positioned on the flat polymer film (14), abutting the latter with its protrusions (5); wherein the container (2) and the flat polymer film (14) are configured to be reversibly attachable to the liquid droplet manipulation instrument (20) in a way that the lower surface (15) of the flat polymer film (14) abuts at least one electrode array (21) of the liquid droplet manipulation instrument (20); and wherein the container (2) is configured to enable displacement of at least one liquid droplet (19) from the at least one well (6) through the channel (12) of the container (2) onto the hydrophobic upper surface (16) of the flat polymer film (14) and above the at least one electrode array (21); the liquid droplet manipulation instrument (20) comprising a control unit (23) with at least one electrode selector (34) connected with at least one voltage control (29), the at least one electrode selector (34) being accomplished to individually select each electrode (35) of the at least one electrode array (21) and provide the selected electrode (35) with a voltage controlled by a voltage control (29) and thus being accomplished to control a guided movement of said liquid droplet (19) on the hydrophobic upper surface (16) of the flat polymer film (14) by electrowetting.
 2. The cartridge (40) of claim 1, wherein the container (2) comprises at least one positioning element (25) for form-fitted attachment of the container (2) to a positioning element (25) of the liquid droplet manipulation instrument (20).
 3. The cartridge (40) of claim 1, wherein the at least one well (6) for positioning a biological sample (9) is sized to accommodate a solid substrate (24) carrying the biological sample (9).
 4. The cartridge (40) of claim 3, wherein the at least one well (6) of the container (2) comprises retention means (31) for preventing the solid substrate (24) to block the opening (11) of said well (6), the retention means (31) being selected from a group comprising a filter, a frit and relief structures on the well's (6) bottom side (8).
 5. The cartridge (40) of claim 1, wherein the cartridge (40) comprises at least one well (6′) that is accomplished as storage for a reaction reagent (10), the stored reaction reagent (10) comprising reagents selected from a group comprising reagents for performing cell lysis, reagents for performing nucleic acid purification, reagents for performing nucleic acid amplification and reagents for performing sequencing of nucleic acids.
 6. The cartridge (40) of claim 1, wherein the protrusions (5) of the container (2) have a height that defines a gap (17) between the hydrophobic upper surface (16) of the film (14) and the base side (4) of the container (2) of less than 2 mm or less than 1 mm when the container (2) is positioned on the flat polymer film (14).
 7. The cartridge (40) of claim 1, wherein the container (2) is made by injection molding of an electrically insulating material (26) and/or an electrically conductive material (27).
 8. The cartridge (40) of claim 1, wherein the container (2) comprises means for identification (30).
 9. The cartridge (40) according to claim 1, wherein the flat polymer film (14) is accomplished as an electrically insulating flat polymer film.
 10. The cartridge (40) according to claim 1, wherein the container (2) and the flat polymer film (14) are accomplished as a separate components that remains to be assembled.
 11. The cartridge (40) of claim 1, wherein the container (2) and the flat polymer film (14) are stably attached to one another.
 12. A kit for enabling droplet manipulation by electrowetting and processing of biological samples in a liquid droplet manipulation instrument (20), the kit including a cartridge (40) according to claim 1 and comprising a container (2) and a flat polymer film (14).
 13. The kit of claim 12, wherein the container (2) and the flat polymer film (14) are accomplished as separate components that remain to be assembled.
 14. The kit of claim 12, wherein the container (2) and the flat polymer film (14) are stably attached to one another.
 15. The kit of claim 12, wherein the flat polymer film (14) is accomplished as an electrically insulating flat polymer film.
 16. A method for processing a biological sample (9) with a liquid droplet manipulation instrument (20), wherein the method comprises the steps of: a) providing a liquid droplet manipulation instrument (20) comprising: at least one electrode array (21) for inducing liquid droplet movements; and a control unit (23) comprising at least one electrode selector (34) connected with at least one voltage control (29), the at least one electrode selector (34) being accomplished to individually select each electrode (35) of the at least one electrode array (21) and provide the selected electrode (35) with a voltage controlled by a voltage control (29); b) providing a container (2) for large volume processing having a top side (3) and a base side (4) and comprising at least one well (6) open at the top side (3) for positioning a biological sample (9) and/or a reaction reagent (10) therein, wherein the at least one well (6) comprises a bottom side (8) having at least one opening (11), the container (2) further comprising a channel (12) connecting the opening of the well (11) with an orifice (13) on the base side (4) of the container (2), and wherein the base side (4) of the container (2) comprises protrusions (5) distributed thereon; and c) providing a flat polymer film (14) having a lower surface (15) and a hydrophobic upper surface (16), which is kept at a distance (d) to the base side (4) of the container (2) by the protrusions (5), the distance (d) defining at least one gap (17) when the container (2) is positioned on the film (14), abutting the latter with its protrusions (5); d) reversibly attaching the container (2) and the film (4) to the liquid droplet manipulation instrument (20), so that the lower surface (15) of the flat polymer film (14) is abutting the at least one electrode array (21); e) displacing at least one liquid droplet (19) from the at least one well (6) through the channel (13) of the container (2) onto the hydrophobic upper surface (16) of the flat polymer film (14) and above the at least one electrode array (21); and f) processing the liquid droplet (19) by a guided movement on the hydrophobic upper surface (16) of the flat polymer film (14) by electrowetting controlled by the liquid droplet manipulation instrument (20).
 17. The method of claim 16, wherein the flat polymer film (14) and the container (2) are provided as separate components that remain to be assembled.
 18. The method of claim 16, wherein the flat polymer film (14) and the container (2) are provided as a cartridge (40) of which the flat polymer film (14) and the container (2) are stably attached to one another.
 19. The method of claim 16, wherein the at least one liquid droplet (19) is moved in the gap (17) within a system liquid that is immiscible with the liquid droplet (19).
 20. The method of claim 18, wherein the cartridge (40) with the flat polymer film (14) and container (2) stably attached to one another is removed from the liquid droplet manipulation instrument (20) and disposed. 